Oncolytic viruses, such as the rabbit-specific myxoma virus (MYXV), represent promising, novel methods to treat patients diagnosed with a variety of solid tumors. While these viruses excel at acutely debulking tumor mass, completely eradicating disease, thus generating durable, complete responses, has proven significantly more challenging. Developing methods to increase the efficacy of oncolytic therapy is therefore of great significance. One of the reasons that oncolytic therapy fails to completely eradicate solid tumors is that these tumors often actively suppress the oncolytically induced immunotherapy which is critical for the therapies success. Our preliminary data demonstrates that this active suppression can be overcome by modifying oncolytic MYXV to secrete a soluble form of the T cell checkpoint protein TIM3. This modification significantly improves the efficacy of MYXV therapy against established tumors and while also displaying reduced toxicity profiles compared to more traditional combination therapy. Unfortunately, the translation of this promising therapy is currently prevented by a lack of understanding concerning the mechanisms involved in treatment which likely cause our current virus to be suboptimal therapeutically. We therefore put forth the current R21 proposal designed to advance the use of our recombinant MYXV into a clinical setting by 1) identifying the specific TIM3:ligand interactions mediating the function of our recombinant virus, and 2) understanding the mechanisms mediating this viruses reduced toxicity profile. The successful completion of this project will have an immediate, positive impact on public health by advancing the use of a novel agent for the treatment of late stage tumors, as well as significant long-term impact by improving our understanding of both MYXV as a therapeutic agent and the potential synergy between oncolytic virotherapy and TIM3-based checkpoint blockade.